Microscopes of the kind recited above, and methods for investigating a sample using such a microscope, are known from practical use and exist in a wide variety of embodiments. In confocal laser microscopy, for example, biological samples are usually labeled with dyes. Different organelles are often labeled with different dyes. These dyes are excited, with an illumination light generated by means of a laser or multiple lasers, to emit light. If multiple dyes are present in the sample, they must usually be depicted on different image channels by means of a wavelength separation in the detection device. The corresponding detected signals pass through detection channels that are settable or embodied for the detection of predefinable different wavelength regions.
The emission spectra of the dyes often overlap. In addition, the reflected excitation light usually falls within the emission spectra of the dyes. Phenomena such as autofluorescence, second harmonic generation, or resonant energy transfer also cannot be distinguished from “normal” fluorescence photons only by wavelength separation. Because these phenomena either furnish additional information about the sample or create an interfering overlay on the pure fluorescence image, it is desirable to be able to separate these phenomena from the actual fluorescence signal in the context of detection.
With (for example, confocal) laser microscopes, the fluorescent light is often chromatically divided prior to detection. This is implemented either via a cascade of optical beam splitters, via a prism, or via an arrangement having a grating. After spectral division the light strikes different detectors in order to form different detection channels. A specific wavelength region is thus associated with each detector or detection channel.
In addition, two methods are known for measurements of fluorescence lifetimes. The first method is single-photon counting, the time between excitation and a detected signal being measured for each photon. Statistics are prepared here over a very large number of individual measurements. The goal here is to ascertain the decay curve and thus the lifetime of dye molecules. Because of the large quantities of data required, evaluation usually occurs offline. A rough estimate of the lifetimes is nevertheless also possible online, by averaging the measured individual values.
A second known method for measuring fluorescence lifetimes is represented by so-called gating methods, in which the photons are sorted into time windows specified before measurement. As with single-photon measurement, statistics are prepared so that the lifetime of the dyes can be calculated after measurement. The known gating methods carry out one measurement for each gating window.